1. Field of the Invention
The present invention relates to a method and composition for the determination of ascorbic acid in aqueous liquids.
2. Description of the Related Art
Ascorbic acid is essential for the normal regulation of the colloidal conditions of intracellular substances, including the fibrils and collagen of connective tissues. Deficiency of Vitamin C (i.e., ascorbic acid) results in abnormal development and maintainence of tissue structures, capillary defects, and eventual development of scurvy. There is, therefore, a need for determining ascorbic acid in body fluids.
Methods and techniques for assaying ascorbic acid are comprehensively reviewed in Rae, J. H., Methods Biochem. Analy. 1, p. 115, 1960 and Henry, R. J., Connor, D. C., and Winkleman, J. W., Clinical Chemistry, Principles and Technics, Second Edition, Harper and Row, 1964, p. 1394. The most commonly used early method involves titration with an oxidation-reduction indicator such as 2,6-dichlorophenolindophenol in acid solution.
Some of the more recent adaptations and approaches to the determination of ascorbic acid are the reduction of mercuric to mercurous chloride by urinary ascorbic acid (Kum-Tatt, L., Leong, P. C., Clin. Chem. 10, p. 575, 1964), resin extraction of urinary ascorbic acid and measurement by 2,6-dichlorindophenol (Hughes, R. E., Analyst, 89, p. 618, 1964), reduction of ferric to ferrous ion and photometric measurement with bathophenanthroline (Vann, L. S., Clin. Chem., 11, p. 979, 1965), reaction with diazotized 4-nitroaniline-2,5-dimethoxyaniline (Michaelsson, G., Michaelsson, M., Scand. J. Clin. Lab. Invest., 20, p. 97, 1967), oxidation of ascorbic acid with 2,6-dichloroindophenol followed by formation of hydrazones (Pelletier, O., J. Lab. Clin. Med., 72, p. 674, 1968), and the reduction of 1,2-naphthoquinone by ascorbic acid to the fluorescent dihydro derivative (Hubmann, B., Monnier, D., Roth, M., Clin. Chim. Acta., 25, p. 161, 1969). Two automated methods have been described. One such method utilizes the production of a dichloroindophenol dye (Garry, P. J., Owen, G. M. Technicon Symp. 1, p. 507, 1967), and the other the 4-methoxy-2-nitroaniline reaction (Wilson, S. S., Guillan, R. A., Clin. Chem., 15, p. 282, 1969).
Most if not all of these prior art techniques are undesirable from two standpoints. First, the selectivity of the various methods has been doubtful because of the relative specificity of the reagents used and the potential that other components of a complex liquid under analysis might be interfering. Second, these methods generally require some or all of the following: extended reaction periods, high degree of technician participation or heating the liquid under analysis to excessively high temperatures on the order of 50.degree.-60.degree. C. which can yield distorted results.
Deutsch, M. J. and Weeks, C. E., Journal of the A.O.A.C. 48, No. 6, 1965, p. 1248 describe the assay of ascorbic acid in foods and vitamin pills using the reaction of o-phenylenediamine with dehydroascorbic acid to form a fluorescent quinoxaline. The dehydroascorbic acid is obtained by treating ascorbic acid with purified charcoal from birch.
There is no suggestion in any of the foregoing publications that the ascorbic acid oxidase interaction can be coupled to the dehydroascorbic acid detection system described by Deutsch et al to yield a highly specific technique for determining ascorbic acid.